A report on the Sixth Molecular Biology of Hearing and Deafness Conference, Hinxton,
UK, 11-14 July 2007.

Meeting report

A meeting held this summer at the Sanger Centre outside Cambridge (UK) focused on
the molecular biology of hearing and deafness. Although several proteins are uniquely
expressed in the vertebrate inner ear, these are primarily found in the sensory hair
cells, where they form key components of the mechanosensitive stereocilia (the ‘hairs’)
of the organ of Corti in the cochlea. In general, inner-ear development is guided
by the same suites of genes that control development in other neurogenic tissues,
making the inner ear a good model system for studying the genetic regulation of development.
In this light, we will focus here on discoveries relating to morphogenesis and cell-fate
determination, the control of gene expression by microRNAs, identification of deafness-associated
genes, and implications of developmental studies for the remediation of hearing loss.

Inner-ear development

Many studies have looked at the overlapping roles of fibroblast growth factors (FGFS)
in inner-ear development. The work on chick presented by Raj Ladher (RIKEN, Kobe,
Japan) was unusual in focusing on the morphogenetic response of the otic placode to
FGF signaling. As might be
predicted from morphogenesis in other tissues, a key component of the transformation
from placode to otic vesicle is a change in cell shape. Ladher reported that FGF signaling
polarizes the distribution of cytoskeletal proteins within the placodal cells, and
that this polarization underlies the shape change. The implications of these results
extend beyond otic placode invagination to gastrulation and neurulation,
two of the most basic morphogenetic events in vertebrate development.

Another key event in ear development is the determination of which cells will become
sensory hair cells and which will become non-sensory supporting cells. A study in
chick by Angelika Dötzlhofer (House Ear Institute, Los Angeles, USA) showed that this
cell-fate determination is somewhat more complex than just toggling the switch for
the ubiquitous Notch signaling pathway, which is involved in determining neural cell
fate in other situations. In particular, she showed that cell fates may be dependent
on the expression of Hes/Hey transcription factors, which can be regulated by both
Notch-dependent and independent mechanisms. As a consequence, sensitivity to disruption
of the Notch signaling pathway may differ between cell types expressing Notch-independent
or Notch-dependent Hes/Hey genes. This differential responsiveness to Notch signaling
might be important to achieve the complex cytoarchitecture of the mature organ of
Corti.

The most striking demonstration that different cell fates can result from differential
expression of the same few genes was presented by Matthew Kelley (National Institute
on Deafness and other Communication Disorders, NIH, Bethesda, USA). In this study,
manipulation of FGF signaling in mice revealed interactions between developing inner
hair cells and the adjacent supporting cells during development. More remarkable was
the demonstration that a separate signal emanates from the Hensen’s cells of the cochlea,
creating a second signaling gradient in the opposite direction to the FGF gradient
emanating from the developing inner hair cells. Thus, there are two cues for position
relative to the cochlear axis, which could account for the radial orientation of stereocilia
bundles and differences along the radial gradient. Previous work by Kelley and colleagues
accounted for the alternation of hair cells and supporting cells in the auditory epithelium;
this new work addresses differentiation
within these cell types and the radial organization and ordering of subtypes. The
new findings could have implications for hair-cell regeneration. Supporting cells,
which often survive traumas that kill sensory hair cells, may have encoded positional
information and might respond accordingly when stimulated to transdifferentiate into
hair cells. Alternatively, the signaling gradients might be constitutively
expressed and cells are oriented by these signals during transdifferentiation.

MicroRNA and the inner ear

New at this year’s meeting was a session on microRNAs (miRNAs), a highly conserved
class of small noncoding RNAs that negatively regulate gene expression by an RNA interference
(RNAi) mechanism. They are produced from primary RNA transcripts by ribonuclease III
family members Drosha and Dicer. Although miRNAs are known to influence cellular proliferation
and differentiation, little is known about their function in mammalian inner-ear development.
Several talks examined the links between miRNA expression, ear development and deafness.

Garret Soukup (Creighton University, Omaha, USA) addressed the function of RNAi and
miRNAs in mouse
inner-ear development using a conditional Dicer knockout. Preliminary results indicate that disruption of the RNAipathway results
in severe morphological defects, suggesting a key role for miRNAs in early inner-ear
development. Donna Fekete (Purdue University, West Lafayette, USA) discussed the treatment
of zebrafish embryos with antisense morpholino oligonucleotides directed against the
miRNAs miR-96, miR-182 and miR-183, which are expressed in the lateral line of zebrafish
and inner-ear sensory epithelium of both mouse and zebrafish. Treatment with these
morpholinos both separately and in combination led to
significant reduction in numbers of hair cells in the anterior and posterior macular
epithelium of the embryonic inner ear without apparent changes in the embryonic stage
or size of the sensory organ. Fekete reported, however, that overexpression of a double-stranded
miR-96 RNA causes an increase in numbers of posterior macular hair cells and the precocious
appearance of hair cells in the sensory cristae of the semicircular canals that detect
head rotation. She suggested that the miRNAs may downregulate the translation of pro-sensory
gene transcripts to facilitate the transition from a pro-sensory state to a hair-cell
state.

MicroRNAs can also be linked to known deafness-related genes. Using bioinformatics
and microarray techniques, Karen Avraham (Tel Aviv University, Tel Aviv, Israel) and
her colleagues screened the mouse genome for predicted miRNA genes located near deafness-related
genes or loci. Assuming
functional conservation during vertebrate evolution, they also looked for murine homologs
of known zebrafish miRNAs with high levels of expression in the zebrafish ear and
lateral line. Avraham reported that some of the candidate deafness-related miRNAs
thus identified in the mouse are differentially expressed over time, or are expressed
differently in the cochlea and the vestibular organs. In addition, they found that
some miRNAs not previously linked to deafness are expressed in the sensory epithelia
of the newborn (postnatal day 0) mouse inner ear. Mutation analysis reported by several
groups at the meeting also implicates miRNAs as critical regulators of mammalian hair-cell
development. It remains unclear what genes are regulated by the miRNAs.

Deafness-related genes in humans

In the past 15 years, hundreds of human genes associated with hereditary deafness
have been identified, and complementary studies in the mouse and zebrafish provide
an avenue for understanding the roles of these genes in ear development and function.
Two new protein-coding genes implicated in human deafness were described at the meeting.
Fatemeh Alasti (National Institute for Genetic Engineering and Biotechnology, Tehran,
Iran, and University of Antwerp, Belgium) reported the identification of a novel homeobox
gene responsible for syndromic microtia (small ear) in an Iranian family. Rob Collin
(Radboud University, Nijmegen, The Netherlands) reported the identification of mutations
in ESRRB (encoding estrogen-receptor related β protein) by genome-wide analysis of single-nucleotide
polymorphisms (SNPs) in a Turkish family and subsequently in the original Pakistani
DFNB35 family, both of which have heritable autosomal recessive non-syndromic deafness.
As reported by several groups in the UK and Australia, mouse N-ethyl-N-nitrosourea
(ENU) mutagenesis screenings are now focused on identification of mutations in a recessive
trait to explore molecules and models for recessive genes of human deafness.

Most genes known to be associated with human deafness have been identified in geographically
isolated populations or small families with monogenic mutations. The elucidation of
genes contributing to complex-trait hearing impairment is in its infancy and is far
behind the investigation of the genetics of other complex diseases. Gene-environment
interactions and ethnicity, age, gender and exclusion criteria (whether related to
other diseases, asymmetric hearing loss, and so on) are all important factors to be
considered. Lut van Laer (University of Antwerp, Belgium) reported that studies of
SNPs in candidate genes found that age-related hearing impairment was associated with
SNPs in the gene for the transcription factor grainyhead-like 2 (GRHL2/DFNA28) in several populations. In addition to the group’s previous findings of an association
of the genes for a voltage-gated potassium channel (KCNE1) and a catalase (CAT) with noise-induced hearing loss, Annelies Konings (University of Antwerp, Belgium)
reported a new association of hearing loss with HSP70 (heat shock protein 70) in two populations.

Hair-cell regeneration

All vertebrates have hair cells, but only in mammals are the hair cells and supporting
cells of the auditory epithelium highly ordered, and only mammals lack the ability
to replace hair cells killed by toxins or loud noise. Several talks addressed the
question of how differences in ear development between mammals and other vertebrates
may account for this lack of regenerative ability.

Bernd Fritzsch (Creighton University, Omaha, USA) pointed out that the decision of
cochlear cells to become hair cells is first indicated by their exiting the cell cycle.
He also argued that Atoh1, a transcription factor that has been the focus of several
hair-cell regeneration studies, is not likely to play a role in that process because
it is not expressed before cell-cycle exit. He also discussed the need for a comprehensive
model of gene regulation during cochlear development in order to understand regeneration,
highlighting the roles of three transcription factors known to be important in normal
development of the chicken and mouse inner ear: Prox1 (innervation), Gata3 (cochlear
elongation) and Lmx1a (cochlear histogenesis).

Michael Lovett (Washington University, St. Louis, USA) presented microarray data from
chickens showing that elements of several signaling pathways known to be important
during inner-ear development are also activated during regeneration after deafening,
including the Notch, Wnt and TGFβ pathways. These results will serve a reference studies
for investigations into whether alterations in these pathways account for the failure
of regeneration after deafening in mature mammals.

After noise or ototoxic chemicals kill hair cells, it is the supporting cells that
must react to the absence of hair cells by sending and responding appropriately to
repair and regeneration signals. Several presentations discussed factors influencing
the ability of supporting cells to respond to the signals they receive during repair
and regeneration. Nicholas Daudet (University College London Ear Institute, London,
UK) reported on the activation of the Notch pathway during hair-cell regeneration
in chickens and speculated that it may preserve a pool of supporting cells for future
repopulation of the depleted auditory epithelium. Jeffrey Corwin (University of Virginia,
Charlottesville, USA) demonstrated that cultures of chick utricular supporting cells
can be perpetuated if they can be induced to separate from the substrate. They will
then form hollow spheres and become polarized, which is critical for the subsequent
differentiation of hair cells in those cultures. Azel Zine (University of Montpellier,
France) described how several types of cochlear supporting cells from the postnatal
mouse could be induced to express stem-cell markers, divide and redifferentiate in
culture.

These results all suggest that cochlear supporting cells can be made receptive to
signals for hair-cell development, but does not explain why they are normally unresponsive.
Yehoash Raphael (University of Michigan, Ann Arbor, USA) illustrated morphological
differences between supporting cells in deafened guinea pigs exposed to different
deafening agents, and also showed that expression of several cell-signaling markers
varies with both deafening agent and time since exposure to the agent. These results
raise the intriguing possibility that the receptiveness of supporting cells to such
developmental signals might depend on the mechanism of deafening and the amount of
time after deafening. The findings may indicate that the failure of supporting cells
to regenerate hair cells in the mammalian ear has different causes under different
circumstances.

Over the past decade we have witnessed a tidal wave of discovery of hundreds of deafness
genes in human families and mouse models and have gained tremendous understanding
of the roles of these genes in inner-ear development and function. This meeting signaled
a new era of hearing research, highlighted by recent discoveries in the contribution
of miRNA, polygenic variations and interactions, molecular signaling, and last, but
not least, increasing hope for treatment for hearing loss and deafness.

Acknowledgements

Conference attendance was supported by NIH NIDCD DC001634 to Y. Raphael (DLS), the
Margaret G. Bertsch Research Endowment (TWG), the Center for Organogenesis University
of Michigan, the Deafness Research Foundation, and the National Organization for Hearing
Research (MM).